4 Genernal Protocol: (For Human Whole Blood) Please Read Important Notes Before Starting The Following Steps. 1. Collect fresh human blood in an anticoagulant-treat collection tube. 2. Add 200~300 μl human whole blood to an appropriately sized micro-centrifuge tube (1.5 ml or 2.0 ml tube). (not provided) --If the sample volume is more than 200 ul, use a 2.0 ml tube as the sample container. 3. Mix 5 volume of RL Buffer with 1 volume of the sample and mix well by inversion. 4. Incubate on ice for 10 min. Vortex briefly 2 times during incubation. 5. Centrifuge for 1 min at 4,500 rpm to form a cell pellet and discard the supernatant completely. 6. Add 600 μl of RL Buffer to resuspend the cell pellet by briefly vortexing. 7. Centrifuge for 1min at 4,500 rpm to form a cell pellet again and discard the supernatant completely. 8. Add 350 µl of FARB Buffer (ß-ME added) to the cell pellet and vortex vigoruusly. Incubate at room temperature for 5 min. (For preparation of FARB Buffer (ß-ME added), See Important Note: 3) 9. Transfer the sample mixture to Filter Column Set and centrifuge at full speed (14,000 rpm or 10,000 x g) for 2 min. 10. Transfer the clarified supernatant from the Collection Tube to a new microcentrifuge tube (not provided), and adjust the volume of the clear lysate. --Avoid to pipette any debris and pellet from the Collection Tube. 11. Add 1 volume of 70% ethanol to the clear lysate and mix well by vortexing. 12. Transfer the ethanol added sample (including any precipitate) to FARB Mini Column Set. Centrifuge at full speed (14,000 rpm or 10,000 x g) for 1 min and discard the flow-through. 3

5 13.(Optional): To eliminate genomic DNA contamination, follow the steps from 13a. Otherwise, proceed to step14 directly. 13a. Add 250 µl of Wash Buffer 1 to wash FARB Mini Column. Centrifuge at full 13b. Add 100 µl of RNase-free DNase 1 solution (0.5U/µl, not provided) to the membrane center of FARB Mini Column. Place the Column on the benchtop for 15 min. 13c. Add 250 µl of Wash Buffer 1 to wash FARB Mini Column. Centrifuge at full 13d. After DNase 1 treatment, proceed to step Add 500 µl of Wash Buffer 1 to wash FARB Mini Column. Centrifuge at full 15. Wash FARB Mini Column twice with 700 µl of Wash Buffer 2 by centrifuge at full --Make sure that ethanol has been added into Wash Buffer 2 when first open. 16. Centrifuge at full speed (14,000 rpm or 10,000 x g) for an additional 3 min to dry the column. --Important Step! This step will avoid the residual liquid to inhibit subsequent enzymatic reaction. 17. Place FARB Mini Column to Elution Tube. 18. Add 50 µl of RNase-free Water to the membrane center of FARB Mini Column. Stand FARB Mini Column for 1 min. --Important Step! For effective elution, make sure that RNase-free Water is dispensed on the membrane center and is absorbed completely. 19. Centrifuge at full speed (14,000 rpm or 10,000 x g) for 2 min to elute RNA. 20. Store RNA at -70ºC. 4

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